BRCA1 Expression Research Articles

Dimethyl Sulfoxide Attenuates Radiation-Induced Testicular Injury through Facilitating DNA Double-Strand Break Repair.

The testis is susceptible to ionizing radiation, and male infertility and sexual dysfunction are prevalent problems after whole-body or local radiation exposure. Currently, there is no approved agent for the prevention or treatment of radiation-induced testicular injury. Herein, we investigated the radioprotective effect of dimethyl sulfoxide (DMSO), an organosulfur compound that acts as a free radical scavenger, on testicular injury. Treatment of mice with a single dose of DMSO prior to 5 Gy irradiation restored sex hormones and attenuated the reduction in testis weight. Histological analyses revealed that DMSO alleviated the distorted architecture of seminiferous tubules and promoted seminiferous epithelium regeneration following irradiation. Moreover, DMSO provided quantitative and qualitative protection for sperm and preserved spermatogenesis and fertility in male mice. Mechanistically, DMSO treatment enhanced GFRα-1+ spermatogonial stem cell and c-Kit+ spermatogonial survival and regeneration after radiation. DMSO also alleviated radiation-induced oxidative stress and suppressed radiation-induced germ cell apoptosis in vivo and in vitro. Additionally, DMSO efficiently reduced DNA damage accumulation and induced the expression of phosph-BRCA1, BRCA1, and RAD51 proteins, indicating that DMSO facilitates DNA damage repair with a bias toward homologous recombination. In summary, our findings demonstrate the radioprotective efficacy of DMSO on the male reproductive system, which warrants further studies for future application in the preservation of male fertility during conventional radiotherapy and nuclear accidents.

Abstract 3311: Darolutamide potentiates the antitumor efficacy of a PSMA-targeted thorium-227 conjugate (PSMA-TTC) in a hormone-independent prostate cancer model

Abstract Androgen receptor (AR) inhibitors are standard of care for the treatment of advanced prostate cancer. Despite an initial response to treatment, patients eventually progress, and novel therapeutic approaches are required. Prostate-specific membrane antigen (PSMA; FOLH1) is an integral membrane glycoprotein that is highly expressed in prostate cancer but has limited expression in normal tissues. PSMA-TTC (227Th-pelgifatamab corixetan; BAY 2315497) consists of the alpha emitter thorium-227 complexed to a 3,2-HOPO chelator conjugated to a PSMA targeting antibody. PSMA-TTC delivers potent radiation to PSMA expressing cells. Here we evaluated the efficacy of PSMA-TTC in combination with darolutamide, a novel AR inhibitor in a hormone insensitive prostate cancer model and investigated the mode of action of the combination treatment. In vitro, darolutamide induced the expression of PSMA in the androgen-independent cell lines C4-2 and 22Rv1 more than 3-fold and 2-fold, respectively. Expression of the apoptosis marker CDKN1A was significantly induced in the PSMA-TTC alone and in the combination group compared to vehicle. Expression of the DNA repair genes BRCA1, XRCC2 and XRCC3 were significantly reduced in PSMA-TTC alone and in the combination group compared to vehicle. In vivo, a single i.v. injection of PSMA-TTC at 300 kBq/kg resulted in a tumor/control (T/C) ratio of 0.44 on day 19 in the 22Rv1 hormone-insensitive prostate cancer model while a twice daily oral treatment with 100 mg/kg darolutamide showed a T/C ratio of 0.82. The combination of darolutamide and PSMA-TTC resulted in a higher efficacy with a T/C ratio of 0.27. Tumor area on day 19 after treatment was significantly reduced in the PSMA-TTC alone group and more so in the combination group, compared to vehicle. As expected, darolutamide alone was not efficacious in this hormone insensitive model. Nine out of 10 mice showed disease control (CR, PR or SD) in the combination group whereas no animals showed disease control in either of the monotherapy groups. This difference was significant by Fisher’s exact test analysis. Treatments were well tolerated with no significant changes in body weight in any of the treatment groups. Using the tumor dissociation kit Miltenyi individual tumor cells were isolated from darolutamide treated and untreated 22Rv1 xenograft tumors. FACS analysis showed a 50fold increase in PSMA expression in the darolutamide treated 22Rv1 tumors compared to vehicle treated animals. These data indicate that darolutamide induces PSMA expression in the hormone independent 22Rv1 model, which may have contributed to the stronger efficacy observed when combined with PSMA-TTC. Altogether these results support the further evaluation of darolutamide combination with PSMA radionuclides. Citation Format: Christoph Schatz, Urs Hagemann, Sabine Zitzmann-Kolbe, Bernard Haendler, Hartwig Hennekes, Stefanie Hammer, Arne Scholz. Darolutamide potentiates the antitumor efficacy of a PSMA-targeted thorium-227 conjugate (PSMA-TTC) in a hormone-independent prostate cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3311.

Abstract 2566: Pre-clinical efficacy of the Wnt pathway inhibitor RXC004 in combination with anti-cancer therapies

Abstract Background: RXC004, a potent and selective inhibitor of the Wnt pathway regulator Porcupine, is being investigated in phase 2 studies in patients with advanced cancers (NCT04907851 and NCT04907539). Wnt pathway alterations, including loss-of-function RNF43 mutations and RSPO gene fusions, result in higher levels of the Wnt receptor Frizzled (Fzd) on the cell surface increasing Wnt-ligand dependent signaling. These alterations, which have been shown to be sensitive to Porcupine inhibitors, are implicated in colorectal, gastric and pancreatic cancer. Pre-clinical in vivo data on the effects of RXC004 in combination with clinically relevant anti-cancer therapies have not previously been described in genetically-defined models of cancer. Methods: RXC004 was evaluated as a monotherapy, and in combination with clinically relevant chemotherapy regimens, in an RSPO-fusion colorectal xenograft model (SNU-1411). RXC004, dosed at 5mg/kg QD (5 days on/2 days off), was evaluated in combination with either the clinically relevant colorectal triplet chemotherapy regimen (5-fluorouracil 5-FU (25mg/kg), irinotecan (50mg/kg) and oxaliplatin (5mg/kg)) or doublet chemotherapy regimen (5-FU (25mg/kg), irinotecan (50mg/kg)). Each chemotherapy regimen was dosed once a week for 3 to 4 weeks either alone or in combination with RXC004, which was dosed from day 8 onwards at 5mg/kg QD (5 days on/2 days off). Efficacy was measured by tumor volume and survival endpoint. In addition, RXC004 was studied in vitro, as monotherapy and in combination with a PARP inhibitor in genetically selected cell lines with both RSPO-fusion and RNF43 mutation. Combination and monotherapy effects on proliferation and effects on downstream signaling were investigated. Results: RXC004 in combination with the triplet chemotherapy regimen led to significantly increased survival compared to either treatment alone (p<0.005) and significant tumor volume reduction measured at Day 22 (p<0.001). RXC004 in combination with the doublet chemotherapy also increased survival compared to either treatment alone (p<0.05), and the combination led to significantly decreased tumor volume measured at Day 25 (p<0.05). RXC004 treatment in genetically defined cell lines with RSPO fusions or RNF43 mutations led to a significant reduction in BRCA1 and BRCA2 gene expression. In in vitro proliferation studies a synergistic effect of RXC004 and a PARP inhibitor was demonstrated in genetically defined cell lines. Conclusion: These data demonstrate that RXC004 in combination with clinically relevant standard of care chemotherapy regimens can lead to potential benefit over chemotherapy alone. The RXC004 induced downregulation of DNA repair pathway genes observed in vitro could contribute to the beneficial effect seen in combination with a PARP inhibitor in vitro as well as the combination effect observed in vivo with standard of care chemotherapy agents. Citation Format: James Kelly, Simon Woodcock, Inder Bhamra, Cliff Jones, Richard Armer, Jane Robertson, Caroline Phillips. Pre-clinical efficacy of the Wnt pathway inhibitor RXC004 in combination with anti-cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2566.

Abstract 216: Expression of DNA damage response proteins modifies the efficacy of CDK4/6 inhibitor-mediated radiosensitization in breast cancer models

Abstract Purpose: CDK4/6 inhibitors (CDK4/6i) are standard of care for the treatment of locally advanced and metastatic estrogen receptor-positive (ER+), HER2-negative metastatic breast cancer (BC). CDK4/6 inhibition + radiation therapy (RT) is synergistic in both ER+ and triple negative breast cancers (TNBC), but the underlying mechanism is not entirely understood. In this study, we evaluated how pre-existing or genetically engineered deficits in DNA damage response genes (BRCA1/2, RAD51, RB1, XRCC6, TP53) influence radiosensitization. We hypothesized that inhibition of homologous recombination (HR) would prevent CDK4/6i-mediated radiosensitization and blocking non-homologous end joining (NHEJ) would be synergistic. Methods: Cellular proliferation assays determined the half-maximal inhibitory concentrations (IC50) of the 3 approved CDK4/6i palbociclib, ribociclib, and abemaciclib. Clonogenic survival assays determined the radiation enhancement ratios (rERs) and evaluated the efficacy of CDK4/6i + RT. Immunofluorescence assays measured RAD51 foci formation and quantified micronuclei formation following RT and/or CDK4/6 inhibition. Immunoprecipitation with myc-RAD51 and GFP-RB assessed potential protein-protein interactions. Results: While ER+ and TNBC cell lines with wild type BRCA1 expression are radiosensitized by CDK4/6i, BRCA1-deficient SUM-149 cells are not radiosensitized by CDK4/6i at concentrations up to 1µM (rER: 0.92-1.01). In an MCF-7 isogenic model of BRCA2 knockout, CDK4/6i-mediated radiosensitization was abolished compared to Cas9 control or parental cell lines. In ER+ BC cell lines (MCF-7-p53 wt, T47D-p53 mutant), transient or genetic knockdown of RAD51 prevented CDK4/6i-induced radiosensitization. The total quantity of RT-induced RAD51 foci increased in vitro following overexpression of RB-a tumor suppressor and downstream target of CDK4/6. RB overexpression also rescued CDK4/6i-mediated radiosensitization in RB-deficient cell lines through changes in HR efficiency but not via NHEJ or altered micronuclei formation. Moreover, immunoprecipitation of RAD51 in ER+ (MCF-7) and TNBC (MDA-MB-231) cells exhibited an interaction with RB. Conversely, loss of the NHEJ-associated protein Ku70 (XRCC6) was synergistic with palbociclib + RT in MCF7 (rER: 1.76-2.44) and T47D (rER: 1.61-3.88) cells. Finally, CRISPR Cas9-mediated loss of the tumor suppressor p53 (TP53) did not affect radiosensitization induced by CDK4/6i in isogenic p53 wt ER+ (MCF-7, rER: 1.19-1.33) and p53 wt TNBC (CAL-51, rER: 1.23-1.52) cell lines with TP53 loss. Conclusions: Taken together, our results in multiple non-overlapping isogenic models of ER+ and TNBC suggest that CDK4/6i-mediated radiosensitization of BC cell lines occurs through impaired HR activity and RB signaling, and not through the actions of p53 or NHEJ-mediated DNA repair. Citation Format: Kassidy M. Jungles, Andrea M. Pesch, Nicole Hirsh, Anna R. Michmerhuizen, Kari Wilder-Romans, Benjamin C. Chandler, Meilan Liu, Lynn Lerner, Lori J. Pierce, James M. Rae, Corey W. Speers. Expression of DNA damage response proteins modifies the efficacy of CDK4/6 inhibitor-mediated radiosensitization in breast cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 216.

Noncanonical NF-κB factor p100/p52 regulateshomologous recombination and modulates sensitivity to DNA-damaging therapy.

Homologous recombination (HR) serves multiple roles in DNA repair that are essential for maintaining genomic stability, including double-strand DNA break (DSB) repair. The central HR protein, RAD51, is frequently overexpressed in human malignancies, thereby elevating HR proficiency and promoting resistance to DNA-damaging therapies. Here, we find that the non-canonical NF-κB factors p100/52, but not RelB, control the expression of RAD51 in various human cancer subtypes. While p100/p52 depletion inhibits HR function in human tumor cells, it does not significantly influence the proficiency of non-homologous end joining, the other key mechanism of DSB repair. Clonogenic survival assays were performed using a pair DLD-1 cell lines that differ only in their expression of the key HR protein BRCA2. Targeted silencing of p100/p52 sensitizes the HR-competent cells to camptothecin, while sensitization is absent in HR-deficient control cells. These results suggest that p100/p52-dependent signaling specifically controls HR activity in cancer cells. Since non-canonical NF-κB signaling is known to be activated after various forms of genomic crisis, compensatory HR upregulation may represent a natural consequence of DNA damage. We propose that p100/p52-dependent signaling represents a promising oncologic target in combination with DNA-damaging treatments.

Targeting nuclear pore complex and therapeutic response in glioblastoma stem cells.

e14000 Background: Glioblastoma is the most prevalent and lethal primary intrinsic tumor in the central nervous system. Glioblastoma stem cells (GSCs) reprogram the transcriptional and metabolic machinery to survive in a dynamic microenvironment. Understanding the regulatory mechanism of GSCs may inform novel therapeutic approaches to improve the clinical outcome for glioblastoma patients. Nuclear pore complex (NPC) is an evolutionarily conserved structure that maintains the traffic between the nucleus and cytoplasm. In current study, we aim to interrogate the aberrant regulation of NPC in GSCs and develop novel targeted approach to sensitize GSC to conventional therapy. Methods: Patient-derived GSCs were validated using functional assays. Transcriptional analysis of GSCs and matched differentiated glioblastoma cells (DGCs) were performed using RNA sequencing to identify aberrant regulatory pathways upregulated in GSCs. The clinical impact of target genes on patient survival was assessed using the glioblastoma dataset from The Cancer Genome Atlas (TCGA). Quantitative RT-PCR and Western blotting were performed to assess the relative expression between GSCs and DGCs. Radiation sensitivity was measured using Cs-137 irradiator. Validation of the stem-like function of target genes was performed using RNA interference followed by in vitro neurosphere formation assay and in vivo tumorigenesis with xenograft mouse model. Results: 27 of the total 31 NPC genes were found to be upregulated in the primary glioblastoma patient specimens compared to non-tumor brain tissue in the TCGA dataset. Among them, a systemic approach combining transcriptional profiling and clinical validation linked nucleoporin 98 (NUP98) to a nexus between GSC phenotype and therapeutic resistance. GSCs preferentially express NUP98, which is essential for GSC maintenance and tumorigenesis in vitro and in vivo. RNA sequencing demonstrated that NUP98 regulates the expression of key DNA repair genes, including BRCA1, BRCA2, and RAD51. NUP98 forms a complex with the transcription factor P65, and directly stimulates the active transcription of genes involved in homologous recombination. Attenuation of NUP98 or P65 expression leads to the accumulation of unrepaired intrinsic DNA damage and sensitizes GSC to the treatment of ionizing radiation. Clinically, overexpression of NUP98 informs poor clinical outcome among patients with glioblastoma. Conclusions: Combining transcriptional discovery and clinical validation, we discovered that NUP98, an essential NPC component, maintains GSC proliferation and tumorigenesis through modulating the transcriptional control of homologous recombination pathway. Our results demonstrated a novel therapeutic approach of sensitizing GSC to conventional treatment through targeting the nexus between aberrant transcriptional regulation and therapeutic resistance in stem-like glioma cells.

Active symptom control with or without oral vinorelbine in patients with relapsed malignant pleural mesothelioma (VIM): A randomised, phase 2 trial

SummaryBackgroundCurrently, there is no US Food and Drug Administration approved therapy for patients with pleural mesothelioma who have relapsed following platinum-doublet based chemotherapy. Vinorelbine has demonstrated useful clinical activity in mesothelioma, however its efficacy has not been formally evaluated in a randomised setting. BRCA1 expression is required for vinorelbine induced apoptosis in preclinical models. Loss of expression may therefore correlate with vinorelbine resistance.MethodsIn this randomised, phase 2 trial, patients were eligible if they met the following criteria: age ≥ 18 years, Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1, histologically confirmed pleural mesothelioma, post platinum-based chemotherapy, and radiological evidence of disease progression. Consented patients were randomised 2:1 to either active symptom control with oral vinorelbine versus active symptom control (ASC) every 3 weeks until disease progression, unacceptable toxicity or withdrawal at an initial dose of 60 mg/m2 increasing to 80 mg/m2 post-cycle 1. Randomisation was stratified by histological subtype, white cell count, gender, ECOG performance status and best response during first-line therapy. The study was open label. The primary endpoint was progression-free survival (PFS), measured from randomisation to time of event (or censoring). Analyses were carried out according to intention-to-treat (ITT) principles. Recruitment and trial follow-up are complete. This trial is registered with ClinicalTrials.gov, number NCT02139904.FindingsBetween June 1, 2016 and Oct 31, 2018, we performed a randomised phase 2 trial in 14 hospitals in the United Kingdom. 225 patients were screened for eligibility, of whom 154 were randomly assigned to receive either ASC + vinorelbine (n = 98) or ASC (n = 56). PFS was significantly longer for ASC+vinorelbine compared with ASC alone; 4.2 months (interquartile range (IQR) 2.2–8.0) versus 2.8 months (IQR 1.4–4.1) for ASC, giving an unadjusted hazard ratio (HR) of 0·60 (80% CI upper limit 0.7, one-sided unadjusted log rank test p = 0.002); adjusted HR 0.6 (80% CI upper limit 0.7, one-sided adjusted log rank test p < 0.001). BRCA1 did not predict resistance to ASC+vinorelbine. Neutropenia was the most common grades 3, 4 adverse events in the ASC +vinorelbine arm.InterpretationVinorelbine plus ASC confers clinical benefit to patients with relapsed pleural mesothelioma who have progressed following platinum-based doublet chemotherapy.FundingThis study was funded by Cancer Research UK (grant CRUK A15569).

The impairment of DDR reduces XBP1s, further increasing DNA damage, and triggers autophagy via PERK/eIF2alpha in MM and IRE1alpha/JNK1/2 in PEL cells

Cancer cells, particularly MM, that are highly secretory cells, and PEL cells that harbor KSHV, are characterized by high level of stress to which they adapt by activating DDR, UPR and autophagy. It is known that UPR sensors may affect DDR, but whether DDR manipulation influences UPR is less known. In this study, we found an intricate interplay between these responses. Indeed, PARP and CHK1 inhibition by AZD2461 and UCN-01, by downregulating c-Myc, reduced the expression of XBP1s, constitutively expressed in these cells, and upregulated CHOP. Interestingly, given the role of XBP1s in regulating DDR, BRCA-1 expression level was reduced, exacerbating DNA damage. Finally, DDR/UPR interplay activated a pro-survival autophagy via PERK/eIF2alpha axis in MM and IRE1alpha/JNK axis in PEL cells, since in the latter case PERK/eIF2alpha activation could be prevented by KSHV that, as other herpesviruses, tries to avoid the blocks of protein translation that this pathway may induce.

Mechanistic Insights into the Hypermethylation of BRCA1 Evinces a Novel Pathway to Breast Tumorigenesis

BackgroundBreast cancer is the most common and dreadful form of cancer globally. Inherited mutations in the breast cancer susceptibility gene, BRCA1, increases the cumulative risk of developing breast cancer by 70‐80%. However, hereditary mutations account for only 5‐10% of the breast cancer burden. At the same time, the rest of this menace is attributed to sporadic cases where patients rarely encounter a BRCA1 mutation. Instead, upto 60% of these patients manifest low BRCA1 expression associated with BRCA1 promoter hypermethylation—moreover, many hereditary cases with a mutated BRCA1 allele exhibit promoter hypermethylation in the second allele. Interestingly, most of these cases also develop into a hyper‐aggressive and drug‐resistant form, Triple Negative Breast Cancer (TNBC), where Estrogen Receptors (ER), Progesterone receptors (PR) and Human epidermal growth factor receptors (Her2) are absent or minimally expressed. But the molecular signaling governing this tumorigenesis has remained elusive with an unresolved question of whether promoter hypermethylation is the master controller of the process of tumorigenesis or is a mere consequence of tumor progression.Objective &amp; MethodologyTo unravel the truth behind this enigma, we induced site‐specific methylations in the CpG island of BRCA1 promoter on wildtype BRCA1 backgrounds using a modified version of CRISPR technology.ResultsInducing site‐specific methylation on the BRCA1 promoter effectuated its downregulation via alteration in the balance between its alternate transcripts ‘β’ and ‘α’. Methylation also attenuated the expression of a long noncoding RNA, NBR2 (Neighbor of BRCA1 gene 2), which is transcribed through the BRCA1 promoter in the reverse direction. It, in turn, activates a feedback loop leading to further downregulation of BRCA1 associated with impaired DNA damage repair and a shift in the balance between apoptosis and autophagy evident under glucose starvation conditions. Further, the β subunit of hCG (human chorionic gonadotrophin) hormone, which is often associated with aggressive forms of BRCA1 mutated breast cancers, was significantly overexpressed. With regards to TNBC progression, methylation also led to the downregulation of the hormone receptors except for ER‐ α, which was found to be overexpressed. This ER‐ α surge helps the cells survive the onslaught following DNA Damage under BRCA1 defective condition.ConclusionFor the first time, this study elucidates the molecular signaling behind breast tumorigenesis involving BRCA1 hypermethylation. Like BRCA1 mutation screening, our findings open up an outstanding opportunity for screening sporadic breast cancers in females using the combination of NBR2, β‐hCG, and BRCA1 hypermethylation as biomarkers from blood. Moreover, as therapeutic intervention, this study also provides scope for the use of BRCA1 hypermethylation reversing drugs like methotrexate, drugs like biguanides that show better action under NBR2 depleted conditions and development of β‐hCG inhibitors for a better prognosis.

BRCA2 C-Terminal RAD51-Binding Domain Confers Resistance to DNA-Damaging Agents.

Breast cancer type 2 susceptibility (BRCA2) protein is crucial for initiating DNA damage repair after chemotherapy with DNA interstrand crosslinking agents or X-ray irradiation, which induces DNA double-strand breaks. BRCA2 contains a C-terminal RAD51-binding domain (CTRBD) that interacts with RAD51 oligomer-containing nucleofilaments. In this study, we investigated CTRBD expression in cells exposed to X-ray irradiation and mitomycin C treatment. Surprisingly, BRCA2 CTRBD expression in HeLa cells increased their resistance to X-ray irradiation and mitomycin C. Under endogenous BRCA2 depletion using shRNA, the sensitivities of the BRCA2-depleted cells with and without the CTRBD did not significantly differ. Thus, the resistance to X-ray irradiation conferred by an exogenous CTRBD required endogenous BRCA2 expression. BRCA2 CTRBD-expressing cells demonstrated effective RAD51 foci formation and increased homologous recombination efficiency, but not nonhomologous end-joining efficiency. To the best of our knowledge, our study is the first to report the ability of the BRCA2 functional domain to confer resistance to X-ray irradiation and mitomycin C treatment by increased homologous recombination efficiency. Thus, this peptide may be useful for protecting cells against X-ray irradiation or chemotherapeutic agents.

Loss of MEN1 function impairs DNA repair capability of pancreatic neuroendocrine tumors.

Somatic MEN1 mutations occur in up to 50% of pancreatic neuroendocrine tumors (PanNETs). Clinical studies have shown that radiation therapy (IR) is effective in a subset of PanNETs, but it remains unclear why some patients respond better to IR than others. Herein, we study whether MEN1 loss of function increases radiosensitivity of PanNETs and determine its effect on DNA double-strand break (DSB) repair. After creating a MEN1 knockout PanNET cell line, we confirmed reduced DSB repair capacity in MEN1-deficient cells and linked these findings to a defect in homologous recombination, as well as reduced BRCA2 expression levels. Consistent with this model, we found that MEN1 mutant cells displayed increased sensitivity to the highly trapping poly (ADP-ribose) polymerase (PARP) 1 inhibitor talazoparib in vitro. Our results suggest that combining IR with PARP inhibition may be beneficial in patients with PanNETs and MEN1 loss of function.

BRCA1: An Endocrine and Metabolic Regulator.

The breast and ovarian cancer susceptibility gene (BRCA1) is a tumor suppressor whose mutation has been associated with the development of breast, ovarian and, probably, other malignancies at young ages. The BRCA1 gene product participates in multiple biological pathways including the DNA damage response, transcriptional control, cell growth and apoptosis. Inactivating germline mutations of the BRCA1 gene can be detected in a substantial portion of families with inherited breast and/or ovarian cancer. While the genomic and cancer-related actions of BRCA1 have been extensively investigated, not much information exists regarding the cellular and circulating factors involved in regulation of BRCA1 expression and action. The present review article dissects the emerging role of BRCA1 as an important regulator of various endocrine and metabolic axes. Experimental and clinical evidence links BRCA1 with a number of peptide and steroid hormones. Furthermore, comprehensive analyses identified complex interactions between the insulin/insulin-like growth factor-1 (IGF1) signaling axis and BRCA1. The correlation between metabolic disorders, including diabetes and the metabolic syndrome, and BRCA1 mutations, are discussed in this article.

Over Karsinomlarında Östrojen, Progesteron Reseptörleri, C-Erb B-2, P53 ve BRCA1’in İmmünhistokimyasal Ekspresyonu ve Prognostik Önemleri

Our study aimed to determine immunohistochemical expression of ER, PR, C-erbB-2, p53 and BRCA1 genes and their relationship with clinicopathological features and prognostic significance. The present study included 85 patients that were diagnosed with epithelial OC, treated and followed-up between 2002 and 2012 at Mersin University, Faculty of Medicine. Immunohistochemical expression of ER, PR, C-erbB-2, p53 and BRCA1 and their relation-ship with clinicopathological parameters were evaluated. In serous carcinomas, expression of ER was significantly associated with metastasis to the fallopian tube, expression of PR was significantly associated with patient’s age, expression of p53 was significantly associated with the progression-free survival (PFS), cytoplasmic expression of BRCA1 was significantly associated with the overall survival (OS) and PFS. In pathogenesis groups, 55 tumors were type 1, and 30 tumors were type 2. There were significant correlations with ER, PR and p53 expressions between the two groups. In our study, PR and p53 were prognostic factors in serous carcinoma. Extensive studies that contain more cases in each histological group are needed to determine the prognostic value of these five immunohistochemical markers.

Expression of BRCA1, BRCA2, RAD51, and other DSB repair factors is regulated by CRL4WDR70

Double-strand break (DSB) repair relies on DNA damage response (DDR) factors including BRCA1, BRCA2, and RAD51, which promote homology-directed repair (HDR); 53BP1, which affects single-stranded DNA formation; and proteins that mediate end-joining. Here we show that the CRL4/DDB1/WDR70 complex (CRL4WDR70) controls the expression of DDR factors. Auxin-mediated degradation of WDR70 led to reduced expression of BRCA1, BRCA2, RAD51, and other HDR factors; 53BP1 and its downstream effectors; and other DDR factors. In contrast, cNHEJ factors were generally unaffected. WDR70 loss abrogated the localization of HDR factors to DSBs and elicited hallmarks of genomic instability, although 53BP1/RIF1 foci still formed. Mutation of the DDB1-binding WD40 motif, disruption of DDB1, or inhibition of cullins phenocopied WDR70 loss, consistent with CRL4, DDB1, and WDR70 functioning as a complex. RNA-sequencing revealed that WDR70 degradation affects the mRNA levels of DDR and many other factors. The data indicate that CRL4WDR70 is critical for expression of myriad genes including BRCA1, BRCA2, and RAD51.

PARP1, BRCA1 and androgen receptor expression in triple-negative breast cancer patients treated with neoadjuvant chemotherapy

IntroductionTriple-negative breast cancer (TNBC) is an aggressive subtype, where no effective therapies have been established for it. Searching for therapeutic targets for TNBC patients is the aim of the current research. Poly adenosine diphosphate ribose polymerase (PARP1) inhibitors are promising antitumor therapy that have a high potency in BRCA1-deficient breast cancers. Materials and methodsForty TNBC patients who received neoadjuvant chemotherapy (NAC) were enrolled in this study, and evaluated for PARP1, BRCA1, and Androgen receptor (AR) immunohistochemical expression before and after receiving NAC. Data of patients' clinical and pathological responses to the chemotherapy were collected and finally analyzed. ResultsThe immunohistochemical results revealed 10 cases (25%) positive for AR, while 18 cases (45%) and 22 cases (55%) expressed PARP1 at low and high levels, respectively. Twelve cases (30%) and 28 cases (70%) expressed BRCA1 at low and high levels, respectively. There was a significant difference between PARP1 expression in normal and malignant tissues (P < 0.001). Higher PARP1 expression was correlated with a better overall clinical response (OAR) and pathological complete response (pCR) (P = 0.018, 0.01 respectively). Co-expression of both PARP1 & BRCA1 was correlated with OAR and pCR. Chemotherapy decreases PARP1 protein levels in matched patient samples (P = 0.015). Positive AR expression was correlated with BRCA1 overexpression. ConclusionPARP1 is highly expressed in TNBC with a better OAR and pCR especially in cases with high BRCA1, so it might be considered as a therapeutic target for this risky group.

Tumor Intrinsic PD-L1 Promotes DNA Repair in Distinct Cancers and Suppresses PARP Inhibitor-Induced Synthetic Lethality.

PD-L1 upregulates BRCA1-mediated homologous recombination, and PD-L1-deficient tumors exhibit BRCAness by manifesting synthetic lethality in response to PARP inhibitors, revealing an exploitable therapeutic vulnerability and a candidate treatment response biomarker. See related commentary by Hanks, p. 2069.

PTEN alterations in sporadic and BRCA1-associated triple negative breast carcinomas

The similarities between sporadic basal-like breast cancer (BLBC) and BRCA1-mutated breast tumours raise the possibility that deregulation of the same pathway may underlie these tumour types. The aim of this study was to determine if PTEN aberrations are characteristic of both BRCA1 tumours and sporadic TN breast carcinomas with low BRCA1 expression, and can thus be used to identify sporadic tumours potentially sensitive to PARP inhibitors. Twelve BRCA1 tumours, 19 non-BRCA familial breast tumours and 71 unselected TN breast carcinomas were screened for PTEN mutations and assessed for PTEN expression and BRCA1 mRNA expression. Loss of PTEN expression was observed in 67% of BRCA1 tumours and more specifically in 89% of TN BRCA1 tumours highlighting the link between PTEN loss and BLBC in the context of germline BRCA1 mutations. Regarding unselected TN tumours, 56% showed PTEN expression loss and 35% displayed low BRCA1 mRNA expression. Unlike familial breast cancers with low BRCA1 mRNA expression, no significant correlation was observed between the loss of PTEN expression and low BRCA1 mRNA expression in this unselected TN tumours panel. Our data suggest that, unlike the germinal context, PTEN and BRCA1 alterations in sporadic TN breast tumours are independent events.

Preferential Involvement of BRCA1/BARD1, Not Tip60/Fe65, in DNA Double-Strand Break Repair in Presenilin-1 P117L Alzheimer Models.

Recently, we showed that DNA double-strand breaks (DSBs) are increased by the Aβ42-amyloid peptide and decreased by all-trans retinoic acid (RA) in SH-SY5Y cells and C57BL/6J mice. The present work was aimed at investigating DSBs in cells and murine models of Alzheimer's disease carrying the preseniline-1 (PS1) P117L mutation. We observed that DSBs could hardly decrease following RA treatment in the mutated cells compared to the wild-type cells. The activation of the amyloidogenic pathway is proposed in the former case as Aβ42- and RA-dependent DSBs changes were reproduced by an α-secretase and a γ-secretase inhibitions, respectively. Unexpectedly, the PS1 P117L cells showed lower DSB levels than the controls. As the DSB repair proteins Tip60 and Fe65 were less expressed in the mutated cell nuclei, they do not appear to contribute to this difference. On the contrary, full-length BRCA1 and BARD1 proteins were significantly increased in the chromatin compartment of the mutated cells, suggesting that they decrease DSBs in the pathological situation. These Western blot data were corroborated by in situ proximity ligation assays: the numbers of BRCA1-BARD1, not of Fe65-Tip60 heterodimers, were increased only in the mutated cell nuclei. RA also enhanced the expression of BARD1 and of the 90 kDa BRCA1 isoform. The increased BRCA1 expression in the mutated cells can be related to the enhanced difficulty to inhibit this pathway by BRCA1 siRNA in these cells. Overall, our study suggests that at earlier stages of the disease, similarly to PS1 P117L cells, a compensatory mechanism exists that decreases DSB levels via an activation of the BRCA1/BARD1 pathway. This supports the importance of this pathway in neuroprotection against Alzheimer's disease.

RB1 loss overrides PARP inhibitor sensitivity driven by RNASEH2B loss in prostate cancer.

Current targeted cancer therapies are largely guided by mutations of a single gene, which overlooks concurrent genomic alterations. Here, we show that RNASEH2B, RB1, and BRCA2, three closely located genes on chromosome 13q, are frequently deleted in prostate cancer individually or jointly. Loss of RNASEH2B confers cancer cells sensitivity to poly(ADP-ribose) polymerase (PARP) inhibition due to impaired ribonucleotide excision repair and PARP trapping. When co-deleted with RB1, however, cells lose their sensitivity, in part, through E2F1-induced BRCA2 expression, thereby enhancing homologous recombination repair capacity. Nevertheless, loss of BRCA2 resensitizes RNASEH2B/RB1 co-deleted cells to PARP inhibition. Our results may explain some of the disparate clinical results from PARP inhibition due to interaction between multiple genomic alterations and support a comprehensive genomic test to determine who may benefit from PARP inhibition. Last, we show that ATR inhibition can disrupt E2F1-induced BRCA2 expression and overcome PARP inhibitor resistance caused by RB1 loss.

Abstract P1-05-06: Establishment of novel BRCA1ness score to quantify BRCAness in breast cancer

Abstract Germline mutations in the loci of BRCA1 or BRCA2 genes with loss of heterozygosity or additional somatic truncation at the WT allele in breast cancer result in characteristic clinicopathological features of homologous recombination deficiency, also known as “BRCAness.”. Although clinical genetic testing for mutations of BRCA1 and BRCA2 has increased the chances of identifying pathogenic variants, we speculated that there may be a difference in cancer biology by the degree of BRCAness. To address this question, we sought to develop a means to estimate the amount of BRCAness using a score consists of BRCA1-mutation-related genes. Thirty-four BRCA1-mutation related genes were selected by high area under the curve (AUC) levels in receiver operating characteristic (ROC) curve between BRCA1 mutation and non-mutation breast cancer, consistently in two large breast cancer cohorts, TCGA and METABRIC. The score was established by using gene set variation analysis (GSVA) algorithm on 34 genes. We found that the BRCA1ness score was the highest in triple negative breast cancer (TNBC) among subtypes consistently in both cohorts (both p &amp;lt; 0.001). Although BRCA1ness score did not correlate with mRNA expression of BRCA1 nor BRCA2 at all (spearman’s rank correlation coefficient (r) = 0.191 and 0.093, respectively), high BRCA1ness score was significantly associated with high rate of BRCA1 mutation (AUC = 0.735 and 0.709, respectively) in both cohorts. This level of AUC for BRCA1ness mutation score was highest compared to other factors, including BRCA1 expression (AUC = 0.323), DNA repair gene set score (AUC = 0.553 and 0.543), nor homologous recombination deficient (HRD) (AUC = 0.659), which are known as the characteristics of BRCAness. Furthermore, the score was significantly correlated with DNA repair gene set score (r = 0.593 and 0.323, both p &amp;lt; 0.01) as well as MKI67 expression (r = 0.655 and 0.626, both p &amp;lt; 0.01) in both cohorts. HRD score was also strongly correlated with BRCA1ness score in the TCGA cohort (r = 0.633). Interestingly, high BRCA1ness was significantly associated with worse disease-free survival (DFS) (p = 0.037 and p &amp;lt; 0.001, respectively) and disease-specific survival (DSS) (p = 0.012 and p &amp;lt; 0.001, respectively) in both cohorts. In conclusion, we established novel BRCA1ness score using mRNA expression of BRCA1-mutation-related genes, and found that it associate with DNA repair and worse survival. Citation Format: Masanori Oshi, Rongrong Wu, Akimitsu Yamada, Li Yan, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe. Establishment of novel BRCA1ness score to quantify BRCAness in breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-06.